Technical Abstract:
Tissue-specific cDNA library sequences (EST) yield a detailed snapshot of gene expression and are useful in developing second-generation molecular resources (i.e. microarrays) for gene expression profiling. We have constructed and characterized BARC-8BOV, a normalized cDNA library developed from intestinal tissues of Holstein dairy cattle synthesized from mRNA isolated from duodenal, jejunal and ileal segments of the small intestines of a lactating dairy cow and neonatal calf. After normalization, there was a significant reduction of high copy number transcripts and a total of 1.8 x 107 clones with an average transcript length of 1,800 base pairs. Sequencing of these clones resulted in 19,110 EST deposited into the NCBI Genbank EST database. Assembly and clustering of these sequences yielded 11,208 unique elements (3,419 contigs and 7,789 singletons) with an average length of 695 base pairs. Analysis strongly suggests normalization and tissue-pooling were effective at increasing the discovery rate of new bovine sequence. A total of 1,123 sequence elements not previously identified in cattle, but with similarity to known genes in other animal systems, were identified and shown to be involved in numerous critical biological processes. An additional 745 transcripts were not represented in nucleotide or protein data bases, and further analysis of these could lead to the identification of gut-specific transcript variants of known genes or potentially the discovery of novel bovine genes. Of the 11,208 assembled sequences, 11,034, or 98.4%, match sequences present in the bovine DNA trace archive at NCBI, and add to a bovine EST database previously lacking significant gut tissue representation. These data will also contribute in efforts to annotate the bovine genome and our aim is to use this library resource to develop second-generation research tools (microarrays, genetic tests, etc.) to understand how gene expression patterns and controls are coupled with cellular, tissue and whole animal nutrient-use profiles. We are particularly interested in identifying genes that regulate or are fundamental to metabolic pathways and growth regulatory processes involved in visceral tissue energy and nutrient use. Ultimately, this work will facilitate development of new nutritional management and selection strategies essential for optimization of animal production.